Single domain antibodies directed against arachidonate 12-lipoxygenase

ABSTRACT

This invention provides compositions and methods to treat a condition or disease without the use of exogenous targeting sequences or chemical compositions. The present invention relates to single-domain antibodies (sdAbs), proteins and polypeptides comprising the sdAbs that are directed against targets that cause a condition or disease. The invention also includes nucleic acids encoding the sdAbs, proteins and polypeptides, and compositions comprising the sdAbs. The invention includes the use of the compositions, sdAbs, and nucleic acids encoding the sdAbs for prophylactic, therapeutic or diagnostic purposes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/249,868 filed on Nov. 2, 2015, and U.S. patentapplication Ser. No. 15/342,044 filed on Nov. 2, 2016, the contents ofwhich are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inASCII text format in lieu of a paper copy. The Sequence Listing isprovided as a file titled “sequence listing.txt,” created Jun. 17, 2019,and is 58 kilobytes in size. The information in the electronic format ofthe Sequence Listing is incorporated herein by reference in itsentirety.

BACKGROUND

The use of single-domain antibodies (sdAbs) as single antigen-bindingproteins or as an antigen-binding domain in larger protein orpolypeptide offers a number of significant advantages over the use ofconventional antibodies or antibody fragments. The advantages of sdAbsinclude: only a single domain is required to bind an antigen with highaffinity and with high selectivity; sdAbs can be expressed from a singlegene and require no post-translational modification; sdAbs are highlystable to denaturing agents or conditions including heat, pH, andproteases; sdAbs are inexpensive to prepare; and sdAbs can accesstargets and epitopes not accessible to conventional antibodies.

There are a number of diseases or conditions, such as viral infectionsor cancer, that are caused by aberrant intracellular or transmembranecomponents such as nucleotides and proteins. Elimination of the aberrantcomponents can be used to prevent or treat the diseases or conditions.There are a number of pharmacological compounds available for treatmentof such diseases, but the compounds can be ineffective, undeliverable,or toxic to unaffected cells.

Other treatments include the use of therapeutic proteins or agents thatcontain an exogenous targeting sequence so that the therapeutic agentcan be recognized by receptors in the cell membrane, enabling thetherapeutic agent to cross the cell membrane and enter the cell. Oncethe therapeutic agent is inside the cell, the therapeutic agent caninteract with the target component in order to treat the disease.However, the use of exogenous targeting sequences can limit the celltype that is targeted by the therapeutic agent, and adds to the cost ofmanufacturing the therapeutic agent.

For the foregoing reasons, there is a need for compositions and methodsto treat or prevent a disease that do not rely on exogenous targetingsequences or chemical compositions in order to enter the cell, and thatare effective in targeting only the affected cells in the body.

The present invention relates to single-domain antibodies (sdAbs),proteins and polypeptides comprising the sdAbs. The sdAbs are directedagainst targets that cause a condition or disease. The invention alsoincludes nucleic acids encoding the sdAbs, proteins and polypeptides,and compositions comprising the sdAbs. The invention includes the use ofthe compositions, sdAbs, proteins or polypeptides for prophylactic,therapeutic or diagnostic purposes. The invention also includes the useof monoclonal antibodies directed towards the sdAbs of the invention.

SUMMARY

The present invention is directed to sdAbs used to treat or prevent acondition or disease. One embodiment is directed to an anti-HumanImmunodeficiency Virus Type 1 (HIV-1) reverse transcriptase singledomain antibody (sdAb). In one aspect, the anti-HIV-1 reversetranscriptase sdAb comprises the amino acid sequence as set forth in SEQID NO:27. The invention also includes a method of treating a disease,preventing development of a disease, or preventing recurrence of adisease in a subject using an anti-HIV-1 reverse transcriptase sdAb byadministration of effective amount of the anti-HIV-1 reversetranscriptase sdAb to a subject in need thereof. The subject can be amammal, such as a human. The anti-HIV-1 reverse transcriptase sdAb canbe administered in combination with one or more compounds such as, forexample, a protease inhibitor. Administration of an effective amount ofthe anti-HIV-1 reverse transcriptase sdAb to a subject in need thereofcan be by intravenous administration, intramuscular administration, oraladministration, rectal administration, enteral administration,parenteral administration, intraocular administration, subcutaneousadministration, transdermal administration, administered as eye drops,administered as nasal spray, administered by inhalation or nebulization,topical administration, and administered as an implantable drug.

In another embodiment, the invention is directed to an isolatedpolypeptide having the amino acid sequence as set forth in SEQ ID NO:27.In another embodiment, the invention includes an antibody directedtoward the polypeptide of SEQ ID NO:27.

It is also contemplated that the invention includes a method ofmeasuring the levels of an anti-HIV-1 reverse transcriptase sdAb in asample from a subject, the method comprising the steps of: a) generatinga mouse monoclonal antibody directed against one or more domains of apolypeptide comprising the amino acid sequence as set forth in SEQ IDNO:27; b) obtaining a sample from the subject; c) performing aquantitative immunoassay with the mouse monoclonal antibody and thesample to determine the amount of sdAb in a subject; thus measuring theamount of sdAb in the subject. In one aspect, the quantitativeimmunoassay comprises an enzyme-linked immunosorbent assay (ELISA),specific analyte labeling and recapture assay (SALRA), liquidchromatography, mass spectrometry, fluorescence-activated cell sorting,or a combination thereof.

Another embodiment of the invention is directed to an anti-Ebola VP24sdAb. In one aspect, the anti-Ebola VP 24 sdAb comprises the amino acidsequence as set forth in SEQ ID NO:55. The invention also includes amethod of treating a disease, preventing development of a disease, orpreventing recurrence of a disease in a subject using an anti-Ebola VP24sdAb by administration of effective amount of the anti-Ebola VP24 sdAbto a subject in need thereof. The subject can be a mammal, such as ahuman. The anti-Ebola VP24 sdAb can be administered in combination withone or more compounds such as, for example, a protease inhibitor.Administration of an effective amount of the anti-Ebola VP24 sdAb to asubject in need thereof can be by intravenous administration,intramuscular administration, oral administration, rectaladministration, enteral administration, parenteral administration,intraocular administration, subcutaneous administration, transdermaladministration, administered as eye drops, administered as nasal spray,administered by inhalation or nebulization, topical administration, andadministered as an implantable drug.

In another embodiment, the invention is directed to an isolatedpolypeptide having the amino acid sequence as set forth in SEQ ID NO:55.In another embodiment, the invention includes an antibody directedtoward the polypeptide of SEQ ID NO:55.

It is also contemplated that the invention includes a method ofmeasuring the levels of an anti-Ebola VP24 sdAb in a sample from asubject, the method comprising the steps of: a) generating a mousemonoclonal antibody directed against one or more domains of apolypeptide comprising the amino acid sequence as set forth in SEQ IDNO:55; b) obtaining a sample from the subject; c) performing aquantitative immunoassay with the mouse monoclonal antibody and thesample to determine the amount of sdAb in a subject; thus measuring theamount of sdAb in the subject. In one aspect, the quantitativeimmunoassay comprises an ELISA, SALRA, liquid chromatography, massspectrometry, fluorescence-activated cell sorting, or a combinationthereof.

Yet another embodiment of the invention is directed to ananti-arachidonate 12-lipoxygenase (ALOX12) sdAb. In one aspect, theanti-ALOX12 sdAb comprises the amino acid sequence as set forth in SEQID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. The inventionalso includes a method of treating a disease, preventing development ofa disease, or preventing recurrence of a disease in a subject using ananti-ALOX12 sdAb by administration of effective amount of theanti-ALOX12 sdAb to a subject in need thereof. The subject can be amammal, such as a human. The anti-ALOX12 sdAb can be administered incombination with one or more compounds. Administration of an effectiveamount of the anti-ALOX12 sdAb to a subject in need thereof can be byintravenous administration, intramuscular administration, oraladministration, rectal administration, enteral administration,parenteral administration, intraocular administration, subcutaneousadministration, transdermal administration, administered as eye drops,administered as nasal spray, administered by inhalation or nebulization,topical administration, and administered as an implantable drug.

In another embodiment, the invention is directed to an isolatedpolypeptide having the amino acid sequence as set forth in SEQ ID NO:49,SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. In another embodiment, theinvention includes an antibody directed toward the polypeptide of SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52.

It is also contemplated that the invention includes a method ofmeasuring the levels of an anti-HIV-1 reverse transcriptase sdAb in asample from a subject, the method comprising the steps of: a) generatinga mouse monoclonal antibody directed against one or more domains of apolypeptide comprising the amino acid sequence as set forth in SEQ IDNO:27; b) obtaining a sample from the subject; c) performing aquantitative immunoassay with the mouse monoclonal antibody and thesample to determine the amount of sdAb in a subject; thus measuring theamount of sdAb in the subject. In one aspect, the quantitativeimmunoassay comprises an ELISA, SALRA, liquid chromatography, massspectrometry, fluorescence-activated cell sorting, or a combinationthereof.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying drawings where:

FIGS. 1 and 2 depict the results of an ELISA using HIV1-9 anti-HIV-1 RTsdAb (SEQ ID NO:27);

FIGS. 3 and 4 depict the results of an ELISA using a dilution series ofHIV1-9 anti-HIV-1 RT sdAb (SEQ ID NO:27);

FIGS. 5 through 8 depict the results of an ELISA using VP24-5 anti-EbolaVP24 sdAb (SEQ ID NO:55); and

FIGS. 9 and 10 depict the results of an ELISA using a dilution series ofVP24-5 anti-Ebola VP24 sdAb (SEQ ID NO:55).

DESCRIPTION

As used herein, the following terms and variations thereof have themeanings given below, unless a different meaning is clearly intended bythe context in which such term is used.

The terms “a,” “an,” and “the” and similar referents used herein are tobe construed to cover both the singular and the plural unless theirusage in context indicates otherwise.

The term “antigenic determinant” refers to the epitope on the antigenrecognized by the antigen-binding molecule (such as an sdAb orpolypeptide of the invention) and more in particular by theantigen-binding site of the antigen-binding molecule. The terms“antigenic determinant” and “epitope” may also be used interchangeably.An amino acid sequence that can bind to, that has affinity for and/orthat has specificity for a specific antigenic determinant, epitope,antigen or protein is said to be “against” or “directed against” theantigenic determinant, epitope, antigen or protein.

As used herein, the term “comprise” and variations of the term, such as“comprising” and “comprises,” are not intended to exclude otheradditives, components, integers or steps.

It is contemplated that the sdAbs, polypeptides and proteins describedherein can contain so-called “conservative” amino acid substitutions,which can generally be described as amino acid substitutions in which anamino acid residue is replaced with another amino acid residue ofsimilar chemical structure and which has little or essentially noinfluence on the function, activity or other biological properties ofthe polypeptide. Conservative amino acid substitutions are well known inthe art. Conservative substitutions are substitutions in which one aminoacid within the following groups (a)-(e) is substituted by another aminoacid within the same group: (a) small aliphatic, nonpolar or slightlypolar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negativelycharged residues and their (uncharged) amides: Asp, Asn, Glu and Gln;(c) polar, positively charged residues: His, Arg and Lys; (d) largealiphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e)aromatic residues: Phe, Tyr and Trp. Other conservative substitutionsinclude: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or intoHis; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly intoAla or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leuinto Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu,into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr;Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ileor into Leu.

A “domain” as used herein generally refers to a globular region of anantibody chain, and in particular to a globular region of a heavy chainantibody, or to a polypeptide that essentially consists of such aglobular region.

The amino acid sequence and structure of an sdAb is typically made up offour framework regions or “FRs,” which are referred to as “Frameworkregion 1” or “FR1”; as “Framework region 2” or“FR2”; as “Frameworkregion 3” or “FR3”; and as “Framework region 4” or “FR4,” respectively.The framework regions are interrupted by three complementaritydetermining regions or “CDRs,” which are referred as “ComplementarityDetermining Region 1” or “CDR1”; as “Complementarity Determining Region2” or “CDR2”; and as “Complementarity Determining Region 3” or “CDR3,”respectively.

As used herein, the term “humanized sdAb” means an sdAb that has had oneor more amino acid residues in the amino acid sequence of the naturallyoccurring VHH sequence replaced by one or more of the amino acidresidues that occur at the corresponding position in a VH domain from aconventional 4-chain antibody from a human. This can be performed bymethods that are well known in the art. For example, the FRs of thesdAbs can be replaced by human variable FRs.

As used herein, an “isolated” nucleic acid or amino acid has beenseparated from at least one other component with which it is usuallyassociated, such as its source or medium, another nucleic acid, anotherprotein/polypeptide, another biological component or macromolecule orcontaminant, impurity or minor component.

The term “mammal” is defined as an individual belonging to the classMammalia and includes, without limitation, humans, domestic and farmanimals, and zoo, sports, and pet animals, such as cows, horses, sheep,dogs and cats.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, PBS (phosphate-bufferedsaline), and 5% human serum albumin. Liposomes, cationic lipids andnon-aqueous vehicles such as fixed oils may also be used. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with a therapeutic agent as defined above, use thereof inthe composition of the present invention is contemplated.

A “quantitative immunoassay” refers to any means of measuring an amountof antigen present in a sample by using an antibody. Methods forperforming quantitative immunoassays include, but are not limited to,enzyme-linked immunosorbent assay (ELISA), specific analyte labeling andrecapture assay (SALRA), liquid chromatography, mass spectrometry,fluorescence-activated cell sorting, and the like.

The term “solution” refers to a composition comprising a solvent and asolute, and includes true solutions and suspensions. Examples ofsolutions include a solid, liquid or gas dissolved in a liquid andparticulates or micelles suspended in a liquid.

The term “specificity” refers to the number of different types ofantigens or antigenic determinants to which a particular antigen-bindingmolecule or antigen-binding protein molecule can bind. The specificityof an antigen-binding protein can be determined based on affinity and/oravidity. The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (KD), is ameasure for the binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the KD, the stronger the binding strength between an antigenicdeterminant and the antigen-binding molecule (alternatively, theaffinity can also be expressed as the affinity constant (KA), which is1/KD). As will be clear to one of skill in the art, affinity can bedetermined depending on the specific antigen of interest. Avidity is themeasure of the strength of binding between an antigen-binding moleculeand the antigen. Avidity is related to both the affinity between anantigenic determinant and its antigen binding site on theantigen-binding molecule and the number of pertinent binding sitespresent on the antigen-binding molecule. Specific binding of anantigen-binding protein to an antigen or antigenic determinant can bedetermined by any known manner, such as, for example, Scatchard analysisand/or competitive binding assays, such as radioimmunoassays (RIA),enzyme immunoassays (EIA) and sandwich competition assays.

As used herein, the term “recombinant” refers to the use of geneticengineering methods (for example, cloning, and amplification) used toproduce the sdAbs of the invention.

A “single domain antibody,” “sdAb” or “VHH” can be generally defined asa polypeptide or protein comprising an amino acid sequence that iscomprised of four framework regions interrupted by three complementaritydetermining regions. This is represented asFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An sdAb of the invention also includes apolypeptide or protein that comprises the sdAb amino acid sequence.Typically, sdAbs are produced in camelids such as llamas, but can alsobe synthetically generated using techniques that are well known in theart. As used herein, the variable domains present in naturally occurringheavy chain antibodies will also be referred to as “VHH domains,” inorder to distinguish them from the heavy chain variable domains that arepresent in conventional 4-chain antibodies, referred to as “VH domains,”and from the light chain variable domains that are present inconventional 4-chain antibodies, referred to as “VL domains.” “VHH” and“sdAb” are used interchangeably herein. The numbering of the amino acidresidues of an sdAb or polypeptide is according to the general numberingfor VH domains given by Kabat et al. (“Sequence of proteins ofimmunological interest,” US Public Health Services, NIH Bethesda, Md.,Publication No. 91). According to this numbering, FR1 of an sdAbcomprises the amino acid residues at positions 1-30, CDR1 of an sdAbcomprises the amino acid residues at positions 31-36, FR2 of an sdAbcomprises the amino acids at positions 36-49, CDR2 of an sdAb comprisesthe amino acid residues at positions 50-65, FR3 of an sdAb comprises theamino acid residues at positions 66-94, CDR3 of an sdAb comprises theamino acid residues at positions 95-102, and FR4 of an sdAb comprisesthe amino acid residues at positions 103-113.

The term “synthetic” refers to production by in vitro chemical orenzymatic synthesis.

The term “target” as used herein refers to any component, antigen, ormoiety that is recognized by the sdAb. The term “intracellular target”refers to any component, antigen, or moiety present inside a cell. A“transmembrane target” is a component, antigen, or moiety that islocated within the cell membrane. An “extracellular target” refers to acomponent, antigen, or moiety that is located outside of the cell.

A “therapeutic composition” as used herein means a substance that isintended to have a therapeutic effect such as pharmaceuticalcompositions, genetic materials, biologics, and other substances.Genetic materials include substances intended to have a direct orindirect genetic therapeutic effect such as genetic vectors, geneticregulator elements, genetic structural elements, DNA, RNA and the like.Biologics include substances that are living matter or derived fromliving matter intended to have a therapeutic effect.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management of adisease or an overt symptom of the disease. The therapeuticallyeffective amount may treat a disease or condition, a symptom of disease,or a predisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve, or affect thedisease, the symptoms of disease, or the predisposition toward disease.The specific amount that is therapeutically effective can be readilydetermined by an ordinary medical practitioner, and may vary dependingon factors known in the art, such as, e.g., the type of disease, thepatient's history and age, the stage of disease, and the administrationof other therapeutic agents.

The present invention relates to single-domain antibodies (sdAbs) thatare directed against viral and intracellular components, as well as toproteins and polypeptides comprising the sdAbs and nucleotides encodingthe proteins and polypeptides. The invention can also relate to sdAbsthat are directed against intercellular, transcellular and extracellulartargets or antigens. The invention also includes nucleic acids encodingthe sdAbs, proteins and polypeptides, and compositions comprising thesdAbs. The invention includes the use of the compositions, sdAbs,proteins or polypeptides for prophylactic, therapeutic or diagnosticpurposes.

SdAbs have a number of unique structural characteristics and functionalproperties which make sdAbs highly advantageous for use as functionalantigen-binding domains or proteins. SdAbs functionally bind to anantigen in the absence of a light chain variable domain, and canfunction as a single, relatively small, functional antigen-bindingstructural unit, domain or protein. This distinguishes sdAbs from thedomains of conventional antibodies, which by themselves do not functionas an antigen-binding protein or domain, but need to be combined withconventional antibody fragments such as antigen-binding fragments (Fab)or single chain variable fragments (ScFv) in order to bind an antigen.

SdAbs can be obtained using methods that are well known in the art. Forexample, one method for obtaining sdAbs includes (a) immunizing aCamelid with one or more antigens, (b) isolating peripheral lymphocytesfrom the immunized Camelid, obtaining the total RNA and synthesizing thecorresponding complementary DNAs (cDNAs), (c) constructing a library ofcDNA fragments encoding VHH domains, (d) transcribing the VHHdomain-encoding cDNAs obtained in step (c) to messenger RNA (mRNA) usingPCR, converting the mRNA to ribosome display format, and selecting theVHH domain by ribosome display, and (e) expressing the VHH domain in asuitable vector and, optionally purifying the expressed VHH domain.

Another method of obtaining the sdAbs of the invention is by preparing anucleic acid encoding an sdAb using techniques for nucleic acidsynthesis, followed by expression of the nucleic acid in vivo or invitro. Additionally, the sdAb, polypeptides and proteins of theinvention can be prepared using synthetic or semi-synthetic techniquesfor preparing proteins, polypeptides or other amino acid sequences.

The sdAbs of the invention will generally bind to all naturallyoccurring or synthetic analogs, variants, mutants, alleles, parts andfragments of the target, or at least to those analogs, variants,mutants, alleles, parts and fragments of the target that contain one ormore antigenic determinants or epitopes that are essentially the same asthe antigenic determinant or epitope to which the sdAbs of the inventionbind in the wild-type target. The sdAbs of the invention may bind tosuch analogs, variants, mutants, alleles, parts and fragments with anaffinity and/or specificity that is the same as, or that is higher thanor lower than the affinity and specificity with which the sdAbs of theinvention bind to the wild-type target. It is also contemplated withinthe scope of the invention that the sdAbs of the invention bind to someanalogs, variants, mutants, alleles, parts and fragments of the targetbut not to others. In addition, the sdAb of the invention may behumanized, and may be monovalent or multivalent, and/or multispecific.Additionally, the sdAbs of the invention can bind to the phosphorylatedform of the target protein as well as the unphosphorylated form of thetarget protein. sdAbs can be linked to other molecules such as albuminor other macromolecules.

In addition, it is within the scope of the invention that the sdAbs aremultivalent, that is, the sdAb can have two or more proteins orpolypeptides which are directed against two or more different epitopesof the target. In such a multivalent sdAb, the protein or polypeptidemay be directed, for example, against the same epitopes, substantiallyequivalent epitopes, or different epitopes. The different epitopes maybe located on the same target, or it could be on two or more differenttargets.

It is also contemplated that the sequence of one or more sdAbs of theinvention may be connected or joined with one or more linker sequences.The linker can be, for example, a protein sequence containing acombination of serines, glycines and alanines.

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of the sdAbs ofthe invention, as long as these are suitable for the described uses.

Since the sdAbs of the invention are mainly intended for therapeuticand/or diagnostic use, they are directed against mammalian, preferablyhuman, targets. However, it is possible that the sdAbs described hereinare cross-reactive with targets from other species, for example, withtargets from one or more other species of primates or other animals (forexample, mouse, rat, rabbit, pig or dog), and in particular in animalmodels for diseases and disorders associated with the disease associatedwith the targets.

In another aspect, the invention relates to a nucleic acid that encodesan sdAb of the invention. Such a nucleic acid may be, for example, inthe form of a genetic construct.

In another aspect, the invention relates to host or host cell thatexpresses or is capable of expressing an sdAb of the invention, and/orthat contains a nucleic acid encoding an sdAb of the invention.Sequences of the sdAbs can be used to insert into the genome of anyorganism to create a genetically modified organism (GMO). Examplesinclude, but are not limited to, plants, bacteria, viruses, and animals.

The invention further relates to methods for preparing or generating thesdAbs, nucleic acids encoding the sdAbs, host cells expressing orcapable of expressing such sdAbs, products and compositions containingthe sdAbs of the invention.

The invention further relates to applications and uses of the sdAbs, thenucleic acids encoding the sdAbs, host cells, products and compositionsdescribed herein. Such a product or composition may, for example, be apharmaceutical composition for treatment or prevention of a disease, ora product or composition for diagnostic use. The sdAbs can be used in avariety of assays, for example ELISA assays and mass spectrometry assaysto measure the serum and tissue levels of the sdAbs.

In another aspect, a nucleic acid encoding one or more sdAbs of theinvention can be inserted into the genome of an organism to treat orprevent diseases.

The present invention generally relates to sdAbs, as well as to proteinsor polypeptides comprising or essentially consisting of one or more ofsuch sdAbs, that can be used for prophylactic, therapeutic and/ordiagnostic purposes.

The methods and compositions detailed in the present invention can beused to treat diseases described herein, and can be used with any dosageand/or formulation described herein or otherwise known, as well as withany route of administration described herein or otherwise known to oneof skill in the art.

The sdAbs of the invention can be used for treatment and prevention ofdiseases caused by viruses or by aberrant cellular proteins. The sdAbsof the present invention can also be used for treatment and preventionof diseases. The sdAbs of the invention can be used to target diseaseswhen there is an overexpression of an intracellular molecule. They canalso be used to treat viral infections by targeting intracellular viralproteins in infected cells. Blocking production of viral proteins, suchas, for example, HIV-1 reverse transcriptase, can block the virallife-cycle.

The sdAbs of the invention can also target intracellular viral proteinssuch as Ebola VP24 and thus block Ebola's ability to shut down thehost's anti-viral immune response.

The sdAbs of the invention can be used with one or more compounds. Forexample, the sdAb of the invention can be used with JAK/STAT inhibitorssuch as, for example, Curcumin, Resveratrol, Cucurbitacin A, B, E, I, Q,Flavopiridol, Deoxytetrangomycin, Cyclopentenone derivatives,N-Acylhomoserine Lactone, Indirubin derivatives, Meisoindigo,Tyrphostins, Platinum-containing compounds (e.g., IS3-295),Peptidomimetics, antisense oligonucleotides, S3I-201, phosphotyrosintripeptide derivatives, HIV protease inhibitors (e.g., nelfinavir,indinavir, saquinavir, & ritornavir), JSI-124, XpYL, Ac-pYLPQTV-NH2, ISS610, CJ-1383, pyrimethamine, Metformin, Atiprimod, S3I-M2001, STX-0119;N-[2-(1,3,4-oxadiazolyl)]-4 quinolinecarboxamide derivative, S3I-1757,LYS;5,8-dioxo-6(pyridin-3-ylamino)-5,8,-dihydro-naphthalene-1-sulfonamide,withacinstin, Stattic, STA-21, LLL-3, LLL12, XZH-5, SF-1066, SF-1087,17o, Cryptotanshinone, FLL32, FLL62, C188-9, BP-1108 and BP-1075,Galiellalactone, JQ1, 5, 15 DPP, WP1066, Niclosamide, SD1008,Nifuroxazide, Cryptotanshinone, BBI quinone, and Ruxolitnib Phosphate.The one or more compounds can increase the therapeutic response andaugment the effectiveness of the sdAbs of the invention. In addition,the effectiveness of the sdAbs can be increased by combining it withpeptides, peptidomimetics, and other drugs, such as, for example, butnot limited to, cimetidine, atorvastatin, celecoxib, metformin, andcimetidine.

It is also contemplated that one or more sdAbs of the invention can becombined, or the sdAbs of the invention can be combined with othersdAbs.

It is contemplated that certain sdAbs of the invention can cross thecell membrane and enter the cell without the aid of additional targetingprotein sequences on the sdAb, and without the aid of exogenouscompounds that direct the sdAb to bind to the cell surface receptors andcross the cell membrane.

After crossing the cell membrane, these sdAbs can target transmembraneor intracellular molecules or antigens. These targets can be, forexample, proteins, carbohydrates, lipids, nucleic acids, mutatedproteins, viral proteins, and prions. The sdAb targets may function asenzymes, structural proteins of the cell, intracellular portions of cellmembrane molecules, molecules within the membranes of organelles, anytype of RNA molecule, any regions of DNA or chromosome, methylated orunmethylated nucleic acids, partially assembled molecules within thesynthesis mechanism of the cell, second messenger molecules, andmolecules within cell signaling mechanisms. Targets may include allmolecules in the cytoplasm, nucleus, organelles, and cell membrane.Molecules destined for secretion or placement in the cell membrane canbe targeted within the cytoplasm before leaving the cell.

The sdAb targets can be in humans, animals, plants, fungi, parasites,protists, bacteria, viruses, prions, prokaryotic cells, and eukaryoticcells. Some examples of intercellular and intracellular signalingmolecules and protein groups that can be targeted by the sdAbs of theinvention are: oncogene products, hormones, cytokines, growth factors,neurotransmitters, kinases (including tyrosine kinase, serine kinase,and threonine kinase), phosphatases, ubiquitin, cyclic nucleotides,cyclases (adenylyl and guanylyl), G proteins, phosphodiesterases, GTPasesuperfamily, immunoglobulins (antibodies, Fab fragments, binders,sdAbs), immunoglobulin superfamily, inositol phosphate lipids, steroidreceptors, calmodulin, CD group (e.g., CD4, CD8, CD28, etc.),transcription factors, TGF-beta, TNF-alpha and beta, TNF ligandsuperfamily, notch receptor signaling molecules, hedgehog receptorsignaling molecules, Wnt receptor signaling molecules, toll-likereceptor signaling molecules, caspases, actin, myosin, myostatin,12-lipoxygenase, 15-lipoxygenase, lipoxygenase superfamily, reversetranscriptase, viruses and their proteins, amyloid proteins, collagen, Gprotein coupled receptors, mutated normal proteins, prions, Ras, Raf,Myc, Src, BCR/ABL, MEK, Erk, Mos, Tp12, MLK3, TAK, DLK, MKK, p38, MAPK,MEKK, ASK, SAPK, JNK, BMK, MAP, JAK, PI3K, cyclooxygenase, STAT1, STAT2,STAT3, STAT4, STAT5a, STAT5b, STAT6, Myc, p53, BRAF, NRAS, KRAS, HRASand chemokines.

HIV is a retrovirus that causes acquired immunodeficiency syndrome(AIDS) in humans. AIDS results in progressive failure of the infectedindividual's immune system, which results in the development oflife-threatening opportunistic infections and cancers. The averagesurvival time after infection with HIV is estimated to be 9 to 11 yearswithout treatment

HIV is transmitted as single-stranded, positive-sense, enveloped RNAvirus. Upon entry into the target cell, the viral RNA genome is reversetranscribed into double-stranded DNA by a virally encoded reversetranscriptase (RT) that is transported along with the viral genome inthe virus particle. RT is an RNA-dependent DNA polymerase and also hasRNaseH activity. The resulting viral DNA is then imported into the hostcell nucleus and integrated into the cellular DNA by a virally encodedintegrase and host co-factors. Once integrated, the virus may becomelatent for months or years. Alternatively, the virus may be transcribed,producing new RNA genomes and viral proteins that are packaged andreleased from the cell as new virus particles.

Two types of HIV have been characterized: HIV-1 and HIV-2. HIV-1 is morevirulent, more infective, and is the cause of the majority of HIVinfections globally. HIV-2 is largely confined to West Africa.

Anti-HIV RT sdAbs were developed to target HIV-1 reverse transcriptase.The anti-HIV-1 RT sdAb may successfully treat individuals infected withHIV either alone or in combination with other retroviral agents. Usingmethods that are well-known in the art, recombinant HIV-1 reversetranscriptase protein (Creative Biomart, Shirley, N.Y.) (SEQ ID NO:1)was used to generate sdAbs that are directed against or can bind to anepitope of HIV-1 RT.

The protein sequence used for immunization of a camel of the recombinantHIV-1 reverse transcriptase protein (SEQ ID NO:1) wasPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICAELEEEGKISRIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSIPLDEDFRKYTAFTIPS TNNETPGTRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYVDDLYVGSDLEIGQHRTKVEELRQHLWRWGFYTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQK

As a result of the immunization, several sdAbs were obtained andscreened. The DNA sequences of the anti-HIV-1 RT sdAbs are listed below:

HIV1-1 (SEQ ID NO: 2):5′-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctgtttacagctacaacacaaactgcatgggttggttccgccaggctccagggaaggagcgcgagggggtcgcagttatttatgctgctggtggattaacatactatgccgactccgtgaagggccgattcaccatctcccaggagaatggcaagaatacggtgtacctgacgatgaaccgcctgaaacctgaggacactgccatgtactactgtgcggcaaagcgatggtgtagtagctggaatcgcggtgaggagtataactactggggccaggggacccaggtcaccgtctcctca-3′ HIV1-2 (SEQ ID NO: 3):5′-caggtgcagctggtggagtctgggggaggctcggtgcaggctggagactctctgagactctcctgtgcagcctctggaaacactgccagtaggttctccatgggctggttccgccaggctccagggaaggagcgcgagggggtcgcggctatttctgctggtggtaggcttacatactatgccgactccgtgaagggccgattcaccatctcccgagacaacgccaagaacacgctgtatctggacatgaacaacctgaaacctgaggacactgccatgtactactgtgccgcaattagtgaccggatgactggtattcaggctcttgcggctctacccagacttcgcccagaagactacggtaactggggccaggggaccctggtcaccgtctcct ca-3′HIV1-7 (SEQ ID NO: 4):5′-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggaccctggtcaccgtct cctca-3′HIV1-8 (SEQ ID NO: 5):5′-caggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggccaggggacccaggtcaccgtct cctca-3′HIV1-6 (SEQ ID NO: 6):5′-caggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccaatatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggccaggggaccctggtcaccgtct cctca-3′HIV1_28 (SEQ ID NO: 7):5′-aggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggccaggggaccctggtcaccgtctc ctca-3′HIV1-21 (SEQ ID NO: 8):5′-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtct cctca-3′HIV1-37 (SEQ ID NO: 9):5′-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtct cctca-3′HIV1-3 (SEQ ID NO: 10):5′-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcactgtct cctca-3′HIV1-5 (SEQ ID NO: 11):5′-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaggcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctaccattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgctaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtct cctca-3′HIV1-10 (SEQ ID NO: 12):5′-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcagcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtct cctca-3′HIV1_29 (SEQ ID NO: 13):5′-gaggtgcagctggtggagtctgggggagactcagtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtct cctca-3′HIV1_32 (SEQ ID NO: 14):5′-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtct cctca-3′HIV1-9 (SEQ ID NO: 15):5′-gaggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctgtttacagctacaacacaaactgcatgggttggttccgccaggctccagggaaggagcgcgagggggtcgcagttatttatgctgctggtggattaacatactatgccgactccgtgaagggccgattcaccatctcccaggagaatggcaagaacacggtgtacctgacgatgaaccgcctgaaacctgaggacactgccatgtactactgtgcggcaaagcgatggtgtagtagctggaatcgcggtgaggagtataactactggggccaggggacccaggtcactgtctcctca-3′ HIV1-16 (SEQ ID NO: 16):5′-caggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctggaaacacctacagtagtagctactgcatgggctggttccgccaggctccagggaaggaccgcgagggggtcgcgcgtattttcactcgaagtggtaccacatactatgccgactccgtgaagggccgattcaccatttcccgtgacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaagacgctgccatgtactactgtgcggcagcccaggggggtgcctgcatttcgtttacttcgttcgcgaagaatttcgtgtaccggggccaggggaccctggtcactgtctcctca-3′ HIV1-13 (SEQ ID NO: 17):5′-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggtcctctgactataactactggggtgaggggaccctggtcaccgtct cctca-3′HIV1_35 (SEQ ID NO: 18):5′-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggtcctctgactataactactggggtgaggggaccctggtcaccgtct cctca-3′HIV1-11 (SEQ ID NO: 19):5′-caggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcactgtct cctca-3′HIV1_22 (SEQ ID NO: 20):5′-caggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtct cctca-3′HIV1-4 (SEQ ID NO: 21):5′-catgtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggaccctggtcaccgtct cctca-3′HIV1_38 (SEQ ID NO: 22):5′-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccaactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactatgactactggggtgaggggaccctggtcaccgtct cctca-3′HIV1_23 (SEQ ID NO: 23):5′-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaagcctctggatacacctacaatagtagagtcgatatcagatctatgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatggacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtct cctca-3′HIV1_25 (SEQ ID NO: 24):5′-gaggtgcagctggtggagtctgggggagactcggtgcaggctggagggtctcttcaactctcctgtaaggcctctggatacacctacaatagtagagtcgatatcagatctgtgggctggttccgccagtatccaggaaaggagcgcgagggggtcgctactattaatattcgtaatagtgtcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacgccctgaaacctgaggacactgccatgtactactgtgcgttgtcagacagattcgcggcgcaggtacctgccaggtacggaatacggccctctgactataactactggggtgaggggacccaggtcaccgtct cctca-3′

The amino acid sequences of the anti-HIV-1 RT sdAbs are shown below:

HIV1-1 (SEQ ID NO: 25): DVQLVESGGGSVQAGGSLRLSCAASVYSYNTNCMGWFRQAPGKEREGVAVIYAAGGLTYYADSVKGRFTISQENGKNTVYLTMNRLKPEDTAMYYCAAKRWCSSWNRGEEYNYWGQGTQVTVSS HIV1-2 (SEQ ID NO: 26):QVQLVESGGGSVQAGDSLRLSCAASGNTASRFSMGWFRQAPGKEREGVAAISAGGRLTYYADSVKGRFTISRDNAKNTLYLDMNNLKPEDTAMYYCAAISDRMTGIQALAALPRLRPEDYGNWGQGTLVTVSSHIV1-9 (SEQ ID NO: 27): EVQLVESGGGSVQAGGSLRLSCAASVYSYNTNCMGWFRQAPGKEREGVAVIYAAGGLTYYADSVKGRFTISQENGKNTVYLTMNRLKPEDTAMYYCAAKRWCSSWNRGEEYNYWGQGTQVTVSS HIV1-16 (SEQ ID NO: 28):QVQLVESGGGSVQAGGSLRLSCAASGNTYSSSYCMGWFRQAPGKDREGVARIFTRSGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDAAMYYCAAAQGGACISFTSFAKNFVYRGQGTLVTVSS HIV1-27 (SEQ ID NO: 29):EVQLGESGGGSVQAGGSLRLSCAASVYSYTTNCMGWFRQAPGKEREGVAVIYSAGGLTYYADSVKGRFTISQDNGKNTVYLTMNRLKPEDTAMYYCAAKRWCSSWNRGEEYNYWGQGTQVTVSS HIV1-30 (SEQ ID NO: 30):QVQLVESGGGSVQAGGSLRLSCAASVYSYNTNCMGWFRQAPGKEREGAAVIYAAGGLTYYADSVKGRFTISQENGKNTVYLTMNRLKPEDTAMYYCAAKRWCSSWNRGEEYNYWGQGTQVTVSS HIV1-21 (SEQ ID NO: 31):EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTQVTVSSHIV1-4 (SEQ ID NO: 32): HVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTLVTVSSHIV1-6 (SEQ ID NO: 33): QVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGQGTLVTVSSHIV1-7 (SEQ ID NO: 34): EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTLVTVSSHIV1-8 (SEQ ID NO: 35): QVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGQGTQVTVSSHIV1-11 (SEQ ID NO: 36): QVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTQVTVSSHIV1-13 (SEQ ID NO: 37): EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRSSDYNYWGEGTLVTVSSHIV1-23 (SEQ ID NO: 38): EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMDALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTQVTVSSHIV1-24 (SEQ ID NO: 39): HVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPGDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGQGTLVTVSSHIV1-25 (SEQ ID NO: 40): EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSVGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTQVTVSSHIV1-31 (SEQ ID NO: 41): DVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTQVTVSSHIV1-38 (SEQ ID NO: 42): EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYANSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYDYWGEGTLVTVSSHIV1-39 (SEQ ID NO: 43): EVQLVESGGDSVQAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPTRYGIRPSDYNYWGQGTQVTVSS

One or more mouse monoclonal antibodies can be generated against one ormore domains of the anti-HIV-1 RT sdAbs of the invention. The mousemonoclonal antibody can be generated by methods that are known by one ofskill in the art, for example, the mouse monoclonal antibody can beproduced by a mouse hybridoma. The mouse monoclonal antibody can be usedin diagnostic assays, for example, the antibody can be used in animmunoassay such as an ELISA or mass spectrometry assay in order tomeasure the amount of anti-HIV-1 RT sdAb present in a sample from apatient.

SdAbs were also generated against a recombinant Arachidonate12-lipoxygenase (ALOX12). ALOX12 is also known as platelet-type12-lipoxygenase, arachidonate oxygen 12-oxidoreductase,Delta12-lipoxygenase, 12Delta-lipoxygenase, C-12 lipoxygenase,leukotriene A4 synthase, and LTA4 synthase. ALOX12 is alipoxygenase-type enzyme that participates in arachidonic acidmetabolism. ALOX12 has been implicated in the development andcomplications of dietary-induced and/or genetically-induced diabetes,adipose cell/tissue dysfunction, and obesity. ALOX12 has also beenthought to regulate blood vessel contraction, dilation, pressure,remodeling, and angiogenesis. Inhibition of ALOX12 prevents thedevelopment of blood vessel formation and thus ALOX12 is a target forreducing neo-vascularization that promotes atherosclerosis,Steatohepatitis, and other arthritic and cancer diseases. Elevatedamounts of ALOX12 may contribute to the development of Alzheimer'sdisease.

The present invention provides sdAbs, proteins, and polypeptides thatare directed against the ALOX12 protein.

It is contemplated that the anti-ALOX12 sdAbs and polypeptides of theinvention can be used for the prevention and/or treatment of diseasesand disorders associated with and/or mediated by ALOX12, such asdiabetes, adipose cell dysfunction, obesity, atherosclerosis,Steatohepatitis, arthritis and cancer.

Recombinant human ALOX12 protein was used to generate sdAbs that aredirected against or can bind to an epitope of ALOX12. To generate theanti-ALOX12 sdAbs, recombinant human ALOX12 was expressed in Escherichiacoli and used as the target antigen.

The recombinant ALOX12 protein sequence (SEQ ID NO:44) used forimmunization of camels was:

MGRYRIRVATGAWLFSGSYNRVQLWLVGTRGEAELELQLRPARGEEEEFDHDVAEDLGLLQFVRLRKHHWLVDDAWFCDRITVQGPGACAEVAFPCYRWVQGEDILSLPEGTARLPGDNALDMFQKHREKELKDRQQIYCWATWKEGLPLTIAADRKDDLPPNMRFHEEKRLDFEWTLKAGALEMALKRVYTLLSSWNCLEDFDQIFWGQKSALAEKVRQCWQDDELFSYQFLNGANPMLLRRSTSLPSRLVLPSGMEELQAQLEKELQNGSLFEADFILLDGIPANVIRGEKQYLAAPLVMLKMEPNGKLQPMVIQIQPPNPSSPTPTLFLPSDPPLAWLLAKSWVRNSDFQLHEIQYHLLNTHLVAEVIAVATMRCLPGLHPIFKFLIPHIRYTMEINTRARTQLISDGGIFDKAVSTGGGGHVQLLRRAAAQLTYCSLCPPDDLADRGLLGLPGALYAHDALRLWEIIARYVEGIVHLFYQRDDIVKGDPELQAWCREITEVGLCQAQDRGFPVSFQSQSQLCHFLTMCVFTCTAQHAAINQGQLDWYAWVPNAPCTMRMPPPTTKEDVTMATVMGSLPDVRQACLQMAISWHLSRRQPDMVPLGHHKEKYFSGPKPKAVLNQFRTDLEKLEKEITARNEQLDWPYE YLKPSCIENSVTI

As a result of the immunization, several sdAbs were obtained andscreened. The DNA sequences of the sdAbs are listed below:

ALOX_21 (SEQ ID NO: 45):5′-gaggtgcagctggtggagtctgggggaggttcggtgcaggctggagggtctctgaggatctcctgtacagcctctggattcacttttgatgacactgacatgggctggtaccgccagactctaggaaatgggtgcgagttggtttctcagattagtaatgatggtagtacattctatagagattccgtgaagggccgattcaccatctcctgggaccgcgtcaacaacacggtgtatctgcaaatgagcgccctgagacctgaggacacggccatgtattactgcaatatcaacgggtgtaggagaccctcgtacaatcttcacttgaacgcatggggccaggggacacaggtcaccgtctcctca-3′ ALOX_41 (SEQ ID NO: 46):5′-caggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgacactgtcctgtgtagcctctggatacggctacagtgccacgtgcatgggctggttccgccaggctccagggaaggagcgcgagggggtcgcgtctatttcaccttatggtgttagaaccttctatgccgactccgcgaaaggccgattcaccgtctcccgagacaacgccaagaacacgctgtatctgcaaatgaacagcctgaaacctgaggacacgtccgtgtactactgtgcggccggttcgggcgttggtgtttgttcactttcgtatccatacacctactggggccaggggacccaggtcaccgtctcctca-3′ ALOX_43 (SEQ ID NO: 47):5′-caggtgcagctggtggagtctgggggaggctcggtgcgggctggagagtctctgagactctcctgtgtagcctctagatccatctatgtttggtactgcatgggctggttccgccaggctgcagggaaggagcgcgagggggtcggaagtatgttcgttggtggcggtaggacatattatgacgactccgtcaagggccgattcaccatctcccaagacaaggccaagaacacgctgtatctgcaaatggacaacctggcacctgaagacactgccatgtattactgtgcggctgggcgctgcggtggcaactggctgagaagcaatgctttcgacaaatggggccaggggacactggtcaccgtctcctca-3′ ALOX_46 (SEQ ID NO: 48):5′-gatgtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagccactggaaacacctacattagccgctgcatgggctggttccgccagcctccagggaaggagcgcgaggtggtcgcacgtatttataccgactctggtaatacatactatcccgacgccgtggagggccgattcaccatctcccaagacaacgccaagaacacgatatatctgcaaatgaacagcctgaaacctgacgacaccgccgtgtactactgtgtgctctcagaggccgtctgtacaaaagaacctggggactttcgttactggggccaggggacccaggtcactgtctcctca-3′

The protein sequences of the anti-ALOX sdAbs generated are as follows:

ALOX_21 (SEQ ID NO: 49): EVQLVESGGGSVQAGGSLRISCTASGFTFDDTDMGWYRQTLGNGCELVSQISNDGSTFYRDSVKGRFTISWDRVNNTVYLQMSALRPEDTAMYYCNINGCRRPSYNLHLNAWGQGTQVTVSS ALOX_41 (SEQ ID NO: 50):QVQLVESGGGSVQAGGSLTLSCVASGYGYSATCMGWFRQAPGKEREGVASISPYGVRTFYADSAKGRFTVSRDNAKNTLYLQMNSLKPEDTSVYYCAAGSGVGVCSLSYPYTYWGQGTQVTVSSALOX_43 (SEQ ID NO: 51): QVQLVESGGGSVRAGESLRLSCVASRSIYVWYCMGWFRQAAGKEREGVGSMFVGGGRTYYDDSVKGRFTISQDKAKNTLYLQMDNLAPEDTAMYYCAAGRCGGNWLRSNAFDKWGQGTLVTVSSALOX_46 (SEQ ID NO: 52): DVQLVESGGGSVQAGGSLRLSCAATGNTYISRCMGWFRQPPGKEREVVARIYTDSGNTYYPDAVEGRFTISQDNAKNTIYLQMNSLKPDDTAVYYCVLSEAVCTKEPGDFRYWGQGTQVTVSS

One or more mouse monoclonal antibodies can be generated against one ormore domains of the anti-ALOX12 sdAbs of the invention. The mousemonoclonal antibody can be generated by methods that are known by one ofskill in the art, for example, the mouse monoclonal antibody can beproduced by a mouse hybridoma. The mouse monoclonal antibody can be usedin diagnostic assays, for example, the antibody can be used in animmunoassay such as an ELISA or mass spectrometry assay in order tomeasure the amount of anti-ALOX12 sdAb present in a sample from apatient.

Ebola, also known as Ebola virus disease (EVD) and Ebola hemorrhagicfever (EHF), is a viral hemorrhagic fever of humans and other primatescaused by Ebolavirus. The disease has a high risk of death, killingbetween 25 and 90 percent of those infected, typically six to sixteendays after symptoms appear.

Ebola interferes with proper functioning of the infected individual'sinnate immune system. Ebola proteins weaken the immune system's responseto viral infections by interfering with the cells ability to produce andrespond to interferon proteins such as interferon-alpha,interferon-beta, and interferon gamma. Ebola's structural proteins, VP24and VP35, play a key role in this interference. The V24 protein blocksthe production of the host cell's antiviral proteins. By inhibiting thehost's immune responses, Ebola quickly spreads throughout the body.

As described herein, anti-VP24 sdAbs were developed to target Ebola'sVP24 protein. The anti-VP24 sdAb may successfully treat individualsinfected with Ebola either alone or in combination with other retroviralagents. Using methods that are well-known in the art, recombinant VP24protein (SEQ ID NO:53) was used to generate sdAbs that are directedagainst or can bind to an epitope of VP24.

The protein sequence recombinant VP24 protein (SEQ ID NO:53) used forimmunization of a camel was:

AKATGRYNLISPKKDLEKGVVLSDLCNFLVSQTIQGWKVYWAGIEFDVTHKGMALLHRLKTNDFAPAWSMTRNLFPHLFQNPNSTIESPLWALRVILAAGIQDQLIDQSLIEPLAGALGLISDWLLTTNTNHFNMRTQRVKEQLSLKMLSLIRSNILKFINKLDALHVVNYNGLLSSIEIILEFNSSLAI

As a result of the immunization, one anti-VP24 sdAb, VP24_5 was obtainedand screened for binding to VP24. The DNA sequence of VP24_5 (SEQ ID.NO:54) is:

5′-ATGGGTGAT GTGCAGCTGGTGGAGTCT GGGGGAGACTCGGTGCGG GCTGGAGGG TCTCTTCAAATGGGTGAT GTGCAGCTGGTGGAGTCT GGGGGAGAC TCGGTGCGGGCTGGAGGGTCTCTTCAACTCTCCTGT AAAGCCTCT GGATACACCTACAATAGTAGAGTCGATATCAGATCT ATGGGCTGG TTCCGCCAGTATCCAGGA AAGGAGCGCGAGGGGGTCGCTACTATT AATATTCGTAATAGTGTC ACATACTAT GCCGACTCCGTGAAGGGCCGATTCACCATCTCCCAA GACAACGCC AAGAACACGGTGTATCTGCAAATGAACGCCCTGAAA CCTGAGGAC ACTGCCATGTACTACTGT GCGTTGTCAGACAGATTCGCGGCGCAG GTACCTGCCAGGTACGGA ATACGGCCC TCTGACTAT AACTACTGG GGTGAGGGGACCCTGGTC ACCGTCTCC TCAAGCTCT GGTCTCGAG-3′

The amino acid sequence of the VP24_5 sdAb (SEQ ID NO:55) is shownbelow, with the CDRs underlined:

MGDVQLVESGGDSVRAGGSLQLSCKASGYTYNSRVDIRSMGWFRQYPGKEREGVATINIRNSVTYYADSVKGRFTISQDNAKNTVYLQMNALKPEDTAMYYCALSDRFAAQVPARYGIRPSDYNYWGEGTLVTVSSSSGLE

One or more mouse monoclonal antibodies can be generated against one ormore domains of the anti-VP24 sdAb of the invention. The mousemonoclonal antibody can be generated by methods that are known by one ofskill in the art, for example, the mouse monoclonal antibody can beproduced by a mouse hybridoma. The mouse monoclonal antibody can be usedin diagnostic assays, for example, the antibody can be used in animmunoassay such as an ELISA or mass spectrometry assay in order tomeasure the amount of anti-VP24 sdAb present in a sample from a patient.

EXAMPLES Example 1: Generation of SdAbs

SdAbs were produced from a camel that was immunized with severalproteins including ALOX12 (SEQ ID NO:44), VP24 (SEQ ID NO:53), and HIV-1reverse transcriptase (SEQ ID NO:1).

Using standard techniques, a phage display library was constructed usingthe pCDisplay-3M vector (Creative Biogene, Shirley, N.Y.) and M13K07helper phage (New England Biolabs, Ipswich, Mass.). Single clones ofsdAbs were confirmed by ELISA, and the DNA and protein sequencesdetermined using standard methods.

Example 2: HIV1-9 (SEQ ID NO:27) SDAB Binds HIV-1 Reverse Transcriptaseand Ebola VP-24

Protein binding experiments were performed on a Biacore 3000 (GeneralElectric Company, Fairfield, Conn.) at 25° C. The assay buffer contained10 mM HEPES buffer (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% P20. Theregeneration buffer contained 10 mM glycine HCl pH 1.75, and theimmobilization buffer contained 10 mM sodium acetate, pH 5.0. The flowrate used for capturing the ligand was 5 ul/min. The flow rate used forkinetics analysis was 30 ul/min.

The ligands used for the protein binding experiment were HIV1-9 (SEQ IDNO:27) and STAT3-VHH 14 (SEQ ID NO:56). The ligands were directlyimmobilized by amine coupling (EDC/NHS) at a response unit (RU) of 1200and 550 on flow cell 2 and 4, respectively, of a CMS sensor chip. Flowcell 1 was kept blank and used for background subtraction. Theun-occupied sites on the CMS chip were blocked with 1M ethanol amine.For binding analysis, the analyte, rHIV-1 (SEQ ID NO:1) was flowed overthe sensor chip. Binding of analyte to the ligand was monitored in realtime. The affinity constant (K_(D)=kd/ka) was calculated from theobserved on rate (ka) of off rate (kd), as shown in Table 1.

The negative control for the protein binding experiments was ananti-STAT3 sdAb,

VHH14 (SEQ ID NO: 56): QVQLVESGGGSVQAGGSLRLSCVASTYTGCMGWFRQAPGKEREGVAALSSRGFAGHYTDSVKGRFSISRDYVKNAVYLQMNTVKPEDAAMYYCAAREGWECGETWLDRTAGGHTYWGQGTLVTVSS

Chi square (χ²) analysis was carried out between the actual sensorgramand the sensorgram generated from the BIAnalysis software to determinethe accuracy of the analysis. A χ² value within 1-2 is consideredaccurate and below 1 is highly accurate.

TABLE 1 Ligand Analyte ka (1/Ms) kd(1/s) Rmax KD (M) Conc. (nM) Chisquare HIV1-9 rHIV-1 8.91 × 10⁴ 3.79 × 10⁻⁴ 71.3 4.25 × 10⁻⁹ 100 0.0321VHH STAT3 rHIV-1 N/A N/A N/A N/A 100 N/A VHH14

Full kinetic analysis was performed at analyte concentrations asindicated in Table 2 with 2 fold serial dilution of the highest analyteconcentration. The HIV1-9 anti-RT sdAb bound both HIV-1 and Ebola VP24analytes.

TABLE 2 Ligand Analyte ka (1/Ms) kd(1/s) Rmax KD (M) Conc. (nM) Chisquare HIV1-9 rHIV-1 1.90 × 10⁵ 7.31 × 10⁻⁴  126 3.85 × 10⁻⁹ 0-200 0.226VHH (1200 RU) STAT3 rHIV-1 NA NA NA NA 0-200 NA VHH14 (550 RU) HIV1-9VP-24 4.38 × 10² 1.66 × 10⁻⁴ 1190 3.79 × 10⁻⁷ 0-200 0.199 VHH (1200 RU)

Example 3: HIV1-9 (SEQ ID NO:27) SDAB Binds HIV-1 Reverse Transcriptasein ELISA

Two different samples of the HIV1-9 anti-HIV-1 RT sdAb (SEQ ID NO:27)was assessed at 1 μg/mL against a checkerboard of coating antigen, 2°antibody and HRP concentrations in an ELISA. The coating antigen wasrecombinant HIV-1 RT (Creative BioMart) (SEQ ID NO:1) at 0.5, 0.025 and0.125 μg/mL per well. The secondary antibody was a rabbit anti-llamabiotinylated diluted at 1:5,000, and 1:10,000, HRP at 1:25,000 and1:50,000. Signal-to-noise ratios>20 were seen with several of theconcentrations. The results of the ELISA are shown in FIGS. 1 and 2.

Three combinations were chosen to assess a dilution series of the HIV1-9anti-HIV-1 RT sdAb (SEQ ID NO:27) (1 μg/mL to 0.0001 μg/mL).

Coating Antigen 2° antibody HRP 0.5 μg/mL 1:10,000 1:25,000 0.5 μg/mL1:5,000 1:50,000 0.5 μg/mL 1:10,000 1:50,000

The results are shown in FIGS. 3 and 4. The two HIV1-9 anti-HIV-1 RTsdAb (SEQ ID NO:27) preparations used have very similar results. Resultswith 0.5 μg/mL coating, 1:5,000 dilution of 2° antibody and 1:50,000dilution of HRP showed binding of HIV1-9 anti-HIV-1 RT sdAb (SEQ IDNO:27) to HIV1 RT (SEQ ID NO:1) with the highest signal-to-noise ratioand a slightly lower blank value.

Example 4: VP24-5 (SEQ ID NO:55) SDAB Binds VP24

Protein binding experiments were performed as described in Example 2.The ligands used for protein binding were VP24-5 (SEQ ID NO:55) andSTAT3-VHH 14 (SEQ ID NO:56). The ligands were directly immobilized byamine coupling (EDC/NHS) at a response unit (RU) of 427 and 550 on flowcell 2 and 4, respectively, of a CMS sensor chip. Flow cell 1 was keptblank and used for background subtraction. The un-occupied sites on theCMS chip were blocked with 1M ethanol amine. For binding analysis, theanalytes, VP24 (SEQ ID NO:53) was flowed over the sensor chip andmonitored in real time. The affinity constant (K_(D)=kd/ka) wascalculated from the observed on rate (ka) of off rate (kd), as shown inTable 3.

TABLE 3 Ligand Analyte ka (1/Ms) kd(1/s) Rmax KD (M) Conc. (nM) Chi2VP24-5- VP-24 1.39 × 10⁵ 8.77 × 10⁻⁴ 6.84 6.31 × 10⁻⁹ 100 0.0481 VHHSTAT3 VP-24 NA NA NA NA 100 NA VHH14

Full kinetic analysis was performed at different analyte concentrationswith 2 fold serial dilution of the highest analyte concentration, asshown in Table 4.

TABLE 4 Ligand Analyte ka (1/Ms) kd(1/s) Rmax KD (M) Conc. (nM) Chi2VP24-5- VP-24 1.61 × 10³ 4.73 × 10⁻⁵ 222 2.94 × 10⁻⁸ 0-200 0.187 VHHSTAT3 VP-24 NA NA NA NA 0-200 NA VHH14 (550 RU)

Example 5: VP24-5 (SEQ ID NO:55) SDAB Binds Ebola VP24 Target in ELISA

Two different samples of the VP24-5 anti-Ebola VP24 sdAb (SEQ ID NO:55)was assessed at 1 μg/mL against a checkerboard of coating antigen, 2°antibody and HRP concentrations in an ELISA. The coating antigen wasrecombinant Ebola VP24 (Creative BioMart) (SEQ ID NO:53) at 0.5, 0.025and 0.125 μg/mL per well. The secondary antibody was a rabbit anti-llamabiotinylated diluted at 1:5,000, and 1:10,000. HRP was used at adilution of 1:10,000 and 1:25,000. The results of the ELISA are shown inFIGS. 5 and 6. The signal-to-noise ratios were low and the analysis wasrepeated with higher concentrations.

The ELISA was repeated with 1 and 0.5 μg/mL VP24-5 anti-Ebola VP24 sdAb(SEQ ID NO:55). Recombinant VP24 (SEQ ID NO:53) was used at either 0.5or 1 μg/mL per well. The secondary antibody was a rabbit anti-llamabiotinylated diluted at 1:1,000, 1:4,000, 1:10,000, and 1:10,000. HRPwas used at a dilution of 1:25,000 and 1:50,000. The results of theELISA are shown in FIGS. 7 and 8.

Three combinations were chosen to assess a dilution series of the VP24-5anti-Ebola VP24 sdAb (SEQ ID NO:55) (1 μg/mL to 0.0001 μg/mL).

Coating Antigen 2° antibody HRP 0.5 μg/mL 1:1,000 1:1,000 0.5 μg/mL1:10,000 1:25,000   1 μg/mL 1:4,000 1:25,000

The results are shown in FIGS. 9 and 10. The two VP24-5 anti-Ebola VP24sdAb (SEQ ID NO:55) preparations used have very similar results, andshow binding of VP24-5 anti-Ebola VP24 sdAb (SEQ ID NO:55) torecombinant VP24 (SEQ ID NO:53).

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments, other embodiments arepossible. The steps disclosed for the present methods, for example, arenot intended to be limiting nor are they intended to indicate that eachstep is necessarily essential to the method, but instead are exemplarysteps only. Therefore, the scope of the appended claims should not belimited to the description of preferred embodiments contained in thisdisclosure. All references cited herein are incorporated by reference intheir entirety.

What is claimed is:
 1. An anti-arachidonate 12-lipoxygenase (ALOX12)single domain antibody (sdAb).
 2. The anti-ALOX12 sdAb of claim 1,wherein the anti-ALOX12 sdAb comprises the amino acid sequence as setforth in SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52.3. A method of treating a disease, preventing development of a disease,or preventing recurrence of a disease in a subject using the anti-ALOX12sdAb according to claim 1, the method comprising administering aneffective amount of the anti-ALOX12 sdAb to a subject in need thereof.4. The method of claim 3, wherein the subject is a mammal.
 5. The methodof claim 4, wherein the mammal is a human.
 6. The method of claim 3,wherein the anti-ALOX12 sdAb is administered in combination with one ormore compounds.
 7. The method of claim 3, wherein administering aneffective amount of the anti-ALOX12 sdAb to a subject in need thereofcomprises intravenous administration, intramuscular administration, oraladministration, rectal administration, enteral administration,parenteral administration, intraocular administration, subcutaneousadministration, transdermal administration, administered as eye drops,administered as nasal spray, administered by inhalation or nebulization,topical administration, and administered as an implantable drug.
 8. Anisolated polypeptide, the isolated polypeptide comprising the amino acidsequence as set forth in SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, orSEQ ID NO:
 52. 9. An antibody directed toward the polypeptide of claim8.
 10. A method of measuring the levels of an anti-ALOX12 sdAb in asample from a subject, the method comprising the steps of: a) generatinga mouse monoclonal antibody directed against one or more domains of apolypeptide comprising the amino acid sequence as set forth in SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52; b) obtaining asample from the subject; c) performing a quantitative immunoassay withthe mouse monoclonal antibody and the sample to determine the amount ofsdAb in a subject; and d) quantifying the amount of sdAb in the subject.11. The method of claim 10 wherein the quantitative immunoassaycomprises an enzyme-linked immunosorbent assay (ELISA), specific analytelabeling and recapture assay (SALRA), liquid chromatography, massspectrometry, fluorescence-activated cell sorting, or a combinationthereof.